In the United States, approximately 12,000 people each year suffer some form of spinal cord injury (SCI), with over 200,000 people chronically paralyzed from SCI. Current therapy for SCI includes surgery, drug treatment and prolonged rehabilitation. However, due to the extensive loss of neural tissue and the poor regenerative capacity of such tissue, the success of current therapy has been limited. The injury of concern is the loss of continuity of bi-directional nerve signals between the brain and the extremities. In most SCIs, the lesioned region of the spinal cord reaches several centimeters in length. Therefore, natural reconnection in these cases is an extremely unlikely event.
Methods for transplantation of neural tissue into the area of the SCI in order to reduce the deficits associated with the injury and to promote functional recovery are currently under development. In animal studies, embryonic tissue transplants into the areas of a lesioned spinal cord have been shown to survive and to reinnervate certain regions of the spinal cord (Bjorklund et al. 1986. Neuroscience 18:685-698; Buchanan and Nornes. 1986. Brain Res. 381:225-236; Moorman et al. 1990. Brain Res. 508:194-198; Ribotta et al. 1996. Brain Res. 707:245-255). Such studies have shown that the time of transplant after injury and the type of cell transplanted affects the success of the attempted transplant. These transplant studies have focused on reinstating nerve fiber connections using ex vivo donor material or attempting to grow long nerve fibers by attractant molecules. However, neither approach to transplantation has had success in growing nerve fibers over a distance of more than a few millimeters.
A variety of methods have been used as a way to bridge or fill spinal cord injury lesions that include transplanting peripheral nerves, transplanting intact fetal spinal cords, transplanting progenitor cells, transplanting stem cells, or transplanting dissociated cells from nervous tissue (McDonald, J. W. 1999. Sci. Amer. 281:64-73; Zompa, E. A. et al. 1997. J. Neurotrauma 14:479-506). Some of these techniques have resulted in improved functional outcome in animal models of spinal cord injury. However, improved function has not been attributed directly to the reinstatement of spinal cord signals through the transplant. Rather, it has been proposed that the primary benefit of the transplanted tissue in these models is through physical and biochemical support for the host tissue surrounding the lesion (Stichel, C. C. and H. W. Muller. 1998. Prog. Neurobiol. 56:119-148; Anderson, D. K. et al. 1995. Brain Pathol. 5:451-457). While the results of these studies have been promising, the goal of re-establishing an axonal connection through a spinal cord lesion has yet to be realized.
Studies have shown that short-term tension on single axon growth cones from chick sensory neurons resulted in xe2x80x9ctowed growthxe2x80x9d (Bray, D. 1984. Develop. Neurobiol. 102:379-389; Lamoureux, P. et al. 1989. Nature 340:159-162; Zheng, J. et al. 1991. J. Neurosci. 11:1117-1125). Though poorly understood, it is believed that this growth mechanism commonly occurs in synapsed CNS axons during embryogenesis and development. Since tracts of synapsed axons have no growth cones from which to extend to match the growth of an organism. Axon elongation must occur from reorganizing and building onto the center length of the axon. It is possible that continuous tensile forces along axons trigger this growth in length.
Elongation of cells used for transplant would therefore be advantageous. Studies with other types of cells have shown that mechanical methods can be used to stretch cells. For example, research on human endothelial cells has shown that mechanical stretching of these cells results in changes in cell orientation and size, as well as cell morphology and function (Yano et al. 1997. J. Cell. Biochem. 64:505-513; Shirinsky et al. 1989. J. Cell Biol. 109:331-339; Galbraith et al. 1998. Cell Motil. Cytoskel. 40:317-330). In one study, mechanical stretching of neuronal cells demonstrated the high tolerance of these cells to dynamic stretch injury (Smith et al. 1999. J. Neurosci. 19:4263-4269). The focus of studies on elongation of cells through mechanical stretching, however, has been on the degree of stretch that can be tolerated before cells lose function or the ability to recover from injury and possible use of these cells in a cell injury model.
It has now been found that mechanically stretched neuronal cells can be produced and used to reconnect damaged spinal cord tissue and reinstate flow of nerve signals.
An object of the present invention is to provide compositions comprising mechanically elongated neuronal cells.
Another object of the present invention is to provide a method for producing elongated cells which comprises culturing selected cells, plating said cultured cells onto an overlying membrane and an underlying membrane so that said cultured cells cover both membranes, and moving the overlying membrane across the underlying membrane via a motor-driven movement so that the cultured cells are mechanically stretched and split into two populations connected by elongated cells. In a preferred embodiment, this method is performed on neuronal cells such as N-tera2 cells.
Yet another object of the present invention is to provide a method for treating nerve injury which comprises transplanting elongated neuronal cells into the nerve of an animal suffering from a nerve at the site of injury. This method would include treatment of spinal cord injury.